The present invention relates to CFTR genes encoding mutant CFTR proteins that result in greater chloride channel activity than the wildtype CFTR gene and protein, when expressed in mammalian cells. These genes can be used advantageously in place of the wildtype CFTR gene for treatment of cystic fibrosis by gene therapy.
Cystic fibrosis (CF) is the most common genetic disease of Caucasians in North America, occurring at a frequency of approximately 1 in 2500 births. Boat et al. (1989) and Welsh et al. (1993) review cystic fibrosis and the molecular basis of the disease. The disease results from defective function of the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein in a variety of tissues, including the pancreas and the lung epithelium. Riordan et al. (1989), Rommens et al. (1989) and Kerem et al. (1989) describe the cloning and sequencing of the CFTR gene. U.S. Pat. No. 5,543,399 to Riordan et al. discloses the purification of CFTR protein.
The absence of CFTR protein in the pancreatic duct results in the blockage of the duct by a thick mucus that prevents pancreatic enzymes from passing from the pancreas to the intestine. Without treatment, CF patients decline as a consequence of malnutrition associated with insufficient pancreatic function. However, pancreatic enzymes may be introduced. into the diet of CF patients as a means of reversing the effects of pancreatic insufficiency.
Unlike in the pancreas, the absence of CFTR function in lung epithelium results in a severe lung disease that cannot be readily reversed or controlled by conventional treatment. Lack of CFTR function in the lung results in airway fluid with an altered ion composition, thereby creating a favorable environment for disease-causing bacteria to colonize the lung. Additionally, mucus secreted into the lung becomes thick and viscous, preventing normal clearing of the bacteria from the airways. The chronic bacterial infection leads to destruction of lung tissue and loss of lung function. Current treatments for CF lung disease involve physical therapy to aid mucus clearance and antibiotic therapy to treat the lung infection. Although these treatments slow the progression of disease, they do not reverse it. Patients with CF consequently die prematurely, usually by the age of 30.
Gene therapy may provide an alternative to conventional therapies for the treatment of cystic fibrosis. CF cells lack CFTR chloride channel activity because they have mutant CFTR genes encoding defective CFTR protein. Gene therapy strategies for the treatment of CF thus involve delivery of a wildtype human CFTR cDNA gene to mutant CF epithelial cells within the lung to restore normal CFTR chloride channel activity. U.S. Pat. No. 5,240,846 to Collin et al. discloses viral and plasmid vectors for CF gene therapy. Rosenfeld et al. (1992), Grubb et al. (1994), Teramoto et al. and Zabner et al. (1993, 1994a and 1994b) describe the use of Adenovirus to transfer the CFTR gene to airway epithelial cells. Gene transfer of the CFTR gene may occur by several different delivery methods. Viral vectors provide an efficient means to deliver the CFTR gene to CF cells, and allow correction of the chloride channel defect in cells infected with recombinant virus containing the wildtype CFTR gene. Recombinant adenovirus containing the wildtype CFTR gene have been shown to efficiently transfer the wildtype CFTR gene. into CF epithelium, and correct the Clxe2x88x92 channel defect. U.S. Pat. No. 5,670,488 discloses Adenovirus vectors for gene therapy. However, high doses of virus are generally required to obtain an efficacious response, and the high doses of virus cause inflammation resulting from the immune response to the viral proteins. Other viruses that may be used for CF gene therapy include AAV (Adeno-associated virus), retrovirus and lentivirus. Flotte et al. (1993) describe the use of AAV in cystic fibrosis gene therapy. The use of these viruses for gene therapy is also limited by immune response to the high titer doses required for an efficacious response.
Gene transfer can also be achieved by transfection of CF cells by lipid-DNA complexes composed of plasmid DNA containing the CFTR cDNA in association with cationic or neutral lipids. Jiang et al. (1998) and Alton et al. (1999) describe the use of cationic lipids for cystic fibrosis gene therapy. Gene therapy utilizing lipid-DNA complexes is a potential alternative to the use of viral vectors and presents a lower risk for an associated inflammatory immune response. However, gene transfer with lipid-DNA complexes is inefficient, so that only a small fraction of cells receive the therapeutic gene. As a consequence, only a very limited correction of the chloride channel defect is possible. Because the efficiency of conventional gene transfer is low, a more substantial correction of the defect would be possible if a CFTR gene were used that was capable of providing higher functional activity than the wildtype CFTR gene. However, there are no currently known means for increasing functional activity of CFTR proteins.
The present invention concerns mutated CFTR genes encoding modified CFTR proteins that result in increased CFTR chloride channel functions in comparison to wildtype CFTR. The modified CFTR proteins of the invention comprise at least one amino acid substitution based on the wildtype CFTR sequence. In a specific embodiment, an amino acid substitution of isoleucine at position 539 to either threonine (I539T) or methionine (I539M) results in increased CFTR chloride channel function, as compared to the wildtype CFTR gene, when expressed in mammalian cells. In another embodiment, glycine at position 550 is substituted with a glutamic acid (G550E). Substitution of amino acids at multiple sites in the CFTR protein is also contemplated in the present invention. The modified CFTR proteins are also an aspect of the subject invention.
The subject invention also concerns methods for increasing CFTR-mediated chloride channel activity in a cell by expressing a polynucleotide encoding a modified CFTR protein of the invention in a target cell.
The subject invention also concerns methods for treating patients with deficiencies in CFTR function. In one embodiment, the modified CFTR genes of the present invention can be used as a therapeutic agent delivered to CF cells by gene therapy. The modified CFTR genes of the present invention provide CFTR proteins with higher CFTR channel activity than that achievable with gene transfer of the wildtype CFTR gene. In a preferred embodiment, the modified CFTR gene encodes a CFTR protein having an amino acid substitution(s) contemplated by the subject invention. Because expression of the modified CFTR protein encoded by the gene results in higher CFTR channel activity, fewer target cells need to be transfected in order to obtain a sufficient correction of the chloride secretion defect.